The present invention is concerned with speciation of organisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish between or among species have not always met the expectations of consumers because they are either too costly, cumbersome or unavailable.
Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among species. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. One assay uses several sets of species-specific primers instead of digestion with restriction enzymes, with identification of the PCR products made by size. Minnick and Barbian, 31 J Microb Meth 51 (1997).
One genus of microorganisms, Bartonella, causes a variety of species-dependent disease states in humans, and is therefore important to speciate prior to treatment. Humans infected with bacteria from the genus Bartonella display a variety of pathogies, and appropriate treatment has been surmised as dependant on the species causing the pathology. For instance, the species B. henselae (relatively common in flea-infested areas) presents as cat scratch disease or atypical cat scratch disease, and health care professionals continue to debate the appropriate antibiotic treatment. Bass et al., 16 Pediatr. Infect. Dis. J., 163 (1997). B. clarridgeiae, another causative agent of cat scratch disease, can be treated with antibiotics, but it is not clear which are the most appropriate. ibid.
B. bacilliformis is the causative agent for Carrion's disease (Oroya fever), and is typically treated with chloramphenicol, penicillins or tetracyclines. ibid. Another species, B. elizabethae has been associated with cardiac valve abnormalities, and is so rare that appropriate antibiotics have yet to be determined. ibid.
B. quintana causes trench fever (rare except for unsanitary living conditions or in the immunocompromised), and has been successfully treated with penicillins, tetracyclines and cephalosporins. Kordick et al., 35(7) J. Clin. Microb. 1813 (1997). B. vinsonii sub vinsonii and B. vinsonii sub berkhoffii have only been found in dogs and voles.
Available Bartonella PCR diagnostics require several steps, and are therefore inconvenient for laboratory analysis of samples. For instance, PCR assays on the basis of differences in citrate synthase sequences have been performed using a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes, followed by gel electrophoresisis to distinguish among species. Joblet et al., 33(7) J. Clin. Microb. 1879 (1995); Norman et al., 33(7) J. Clin. Microb. 1797 (1995). PCR assays on the basis of differences in 16S rRNA sequences have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129 FEMS Microbiol. Letters 261 (1995).
Likewise, primers have been used to amplify the region between the 16S and 23S genes (called "the intergenic region") of Bartonella. In those assays, restriction enzymes were also used to cut and distinguish the PCR products. Matar et al., 31(7) J. Clin. Microb. 1730 (1993) and Roux and Raoult, 33(6) J. Clin. Microb. 1573 (1995). In Roux, a difference in size of PCR products (prior to digestion by restriction enzymes) was noted (page 1576); however, the differences are so small as to be indistinguishable on a gel. Moreover, no suggestion is made in Roux to use the pre-digestion PCR product size differences for the purpose of differentiation. In Matar, page 1732 that all three species had "an approximately 1,600-bp fragment" and bacilliformis had a 1,000 bp fragment prior to digestion.
In a different approach, Minnick and Barbian, 31 J Microb Meth 51 (1997) designed one set of primers from the 16S/23S intergenic region of Bartonella, and amplified fragments from B. bacilliformis, B. elizabethae, B. henselae and B. vinsonii. The fragments were of different, but indistinguishable sizes (FIG. 2), and the researchers therefore conducted a second, species-specific amplification using different sets of primers for each species represented (FIG. 3). Minnick, at 55 (1997).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.